Vol. 84: 237–242, 2009 DISEASES OF AQUATIC ORGANISMS Published April 27 doi: 10.3354/dao02046 Dis Aquat Org

Microsporidiosis in the Caribbean spiny argus from southeast Florida, USA

Yasunari Kiryu1,*, Donald C. Behringer2, Jan H. Landsberg1, Barbara D. Petty2, 3

1Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 Eighth Avenue SE, St. Petersburg, Florida 33701, USA 2Program in Fisheries and Aquatic Sciences, School of Forest Resources and Conservation, University of Florida, 7922 NW 71 Street, Gainesville, Florida 32653, USA 3Large Clinical Sciences, College of Veterinary Medicine, 7922 NW 71 Street, University of Florida, Gainesville, Florida 32653, USA

ABSTRACT: Two specimens of the Caribbean captured by lobster fish- ers offshore of southeast Florida, USA, between late 2007 and early 2008 had completely white abdominal muscle tissue with a ‘cooked’ appearance. Wet-mount examination of the skeletal muscle tissue revealed masses of microsporidian spores. Histopathology of longitudinally sectioned skeletal muscle showed that the microsporidian spores displaced much of the muscle mass, but were inter- spersed with small empty vacuoles (approximately 5 µm in diameter) found adjacent to necrotic skeletal muscle. Skeletal muscle showed both liquefactive and coagulative necrosis. Transmission electron microscopy of the microsporidian spores revealed characteristics — including microvilli extending from the surface of the exospore, a unikaryotic spore (width 1.0 ± 0.13 µm, range 0.8 to 1.4 µm; length 1.4 ± 0.11 µm, range 1.2 to 1.6 µm; mean ± SD, N = 16), and an isofilar polar filament — consistent with the genus Ameson, which is known to infect other palinurid . Microsporidio- sis in Caribbean spiny lobsters has rarely been reported within the lobster’s range, with only one brief report coming from the Florida Keys in 1976. Potential risks to the lobster fishery are unknown but warrant further study.

KEY WORDS: Microsporidia · Infection · Parasite · Spiny lobster · Panulirus argus · Skeletal muscle · Necrosis · Transmission electron microscopy

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INTRODUCTION et al. 1977, Azevedo 1987, Olson et al. 1994, Lightner 1996, Azevedo et al. 2000, Wang & Chen 2007). Micro- Spiny lobsters have few reported diseases (Evans et al. sporidia usually infect the skeletal musculature, causing 2000); however, with continued interest in their culture mechanical damage and altering the biochemical and grow-out, increased health problems are anti- composition of the host tissue (Findley et al. 1981). The cipated. Microsporidian parasites have been reported combined effect changes the coloration of normal muscle to infect palinurid lobsters and are potentially patho- tissue from translucent gray to cottony-white. genic to wild stocks (Bach & Beardsley 1976, Evans et The possible effects of microsporidiosis on crus- al. 2000). The phylum Microsporidia is currently classi- tacean fisheries and aquaculture are considerable, in- fied within the kingdom Fungi, but their taxonomic cluding the reduced marketability of cultured penaeid status is under debate (Hibbett et al. 2007). Micro- (Lightner 1996). with micro- sporidiosis in decapod crustaceans is caused by several sporidiosis are not aesthetically pleasing, and the mus- genera of parasitic microsporidia, including Abelspora, cle is in poor condition for the market, thereby reduc- Agmasoma, Ameson, Endoreticulatus, Gurleya, Ino- ing its commercial value. dosporus, Nadelspora, Nosema, Ormieresia, Pleisto- Although there is a known history of microsporidio- phora and Thelohania (Sprague & Couch 1971, Vivarès sis in decapods in Florida, e.g. Agmasoma duorara in

*Email: [email protected] © Inter-Research 2009 · www.int-res.com 238 Dis Aquat Org 84: 237–242, 2009

wild pink shrimp Farfantepenaeus duorarum (Iversen resin (at FWRI). Slides were stained with hematoxylin & Manning 1959, Kruse 1959), microsporidiosis in the and eosin (H&E), periodic acid Schiff/ metanil yellow Caribbean spiny lobster Panulirus argus is poorly stud- (PAS-MY) and thionin (Quintero-Hunter et al. 1991), ied (Shields et al. 2006). To our knowledge, the only and examined histopathologically using light micro- brief report (Bach & Beardsley 1976) did not identify scopy. Light photomicrographs were captured with a the specific parasite. The present report presents the microscope (Olympus BX51) equipped with a digital results of pathological investigations of 2 Caribbean camera (Olympus DP71). A smaller piece of the skele- spiny lobster specimens — one collected by a recre- tal muscle (approximately 1 × 1 × 1 mm) from each lob- ational lobster fisher offshore of Pompano Beach, ster was processed for transmission electron micro- Florida (approximately 26° 13’ N, 80° 01’ W), on scopic (TEM) examination. At FWRI, the tissue was December 17, 2007, and another collected by a recre- fixed overnight in Trumps fixative (4% formaldehyde, ational fisher offshore of Jupiter, Florida (approxi- 1% glutaraldehyde, 50 mM NaH2PO4, pH 7.2) fol- mately 26° 55’ N, 80° 01’ W), on March 31, 2008. After lowed by post-fixation in 1% osmium tetroxide (OsO4) removing the carapace, the fishers noticed an unusual for 1 h. Tissues were subsequently dehydrated in a white color to the abdominal skeletal muscle tissue. graded ethanol series, infiltrated with epoxy propylene Although these 2 reports may have just been coinci- oxide and embedded in epoxy resin. The epoxy block dental, considering that they came shortly after the dis- was then sectioned with an EM UC6 ultramicrotome covery of P. argus Virus 1 (PaV1), the first naturally (Leica Microsystems), stained with uranyl acetate fol- occurring viral pathogen found infecting any lobster lowed by lead citrate, and examined with a TEM (Shields & Behringer 2004), further investigation was (JEM-1400, JEOL) equipped with a digital camera warranted. PaV1 has been well described in the (ORIUSTM SC1000 CCD, GATAN). By capturing digi- Florida Keys (Behringer et al. 2006, 2008, Butler et al. tized TEM images with an embedded scale bar, the 2008) and along the Caribbean coast of Mexico approximate spore size was determined by measuring (Huchin-Mian et al. 2008). Given the importance of P. maximum widths and heights (µm) of randomly argus to recreational and commercial fisheries and the selected mature pyriform spores (N = 16). The epoxy increased awareness of disease generated by reports block was also thick-sectioned (1 µm) and stained with of PaV1, we anticipate that fishers will more likely toluidine blue for confirmation of spores by light report incidences of microsporidiosis if it is indeed microscopy. At the University of Florida, the tissue for increasing in prevalence and emerging as a pathogen TEM examination was processed by the Electron of concern.

MATERIALS AND METHODS

The abdomen of the Pompano Beach Panulirus argus specimen, including the shell and skeletal muscle, was frozen and later shipped to the University of Florida in Gainesville. The same tissue from the Jupiter specimen was shipped overnight on ice within 24 h of collection to the Florida Fish and Wildlife Conser- vation Commission’s Fish and Wildlife Research Institute (FWRI), St. Peters- burg, Florida. Upon arrival at each in- stitution, the condition of the lesioned tissue was documented with digital ma- crophotographs and assessed using wet- mount light microscopy. Skeletal muscle was also smeared onto a glass slide and stained with a Diff-Quick stain kit (IMEB). Small sections of skeletal muscle (10 × 5 × 5 mm) were fixed in 10% Fig. 1. Panulirus argus. Specimen from the Jupiter site. (A) Abdomen and telson buffered formalin, embedded in either with shell attached and the white-discolored skeletal muscle seen at the top. (B) paraffin (at University of Florida) or JB-4 White-discolored skeletal muscle of abdomen with shell and telson removed Kiryu et al.: Microsporidiosis in Panulirus argus 239

Microscopy BioImaging Laboratory using a laboratory chalky appearance (Fig. 1). The texture of the muscle microwave (PELCO® BioWave with ColdSpot, Ted from the fresh Jupiter specimen was relatively firm. Pella). Microwave settings were as follows: sample The muscle tissue from the frozen and thawed Pom- temperature restricted to 37°C using a ColdSpot tem- pano specimen appeared flaccid, but this texture was perature probe, vacuum set at 22 bars, and microwave probably due to freezing. power set at 180 W. Tissue was fixed in Trump’s fixa- Initial wet-mount light microscopy of the skeletal tive for 45 s under vacuum, washed 3 times with 0.1 M muscle tissue revealed masses of single-celled spores sodium cacodylate buffer for 45 s each time; postfixed (Fig. 2). The sporophorous vesicles (SPV or sporont) with 2% buffered OsO4 under vacuum (1 min at room typically associated with Thelohania or Agmasoma temperature [RT], 45 s microwave [MW], 3 min RT), spores were not observed. Slides stained with Diff- washed twice with water for 45 s each time, and dehy- Quick revealed spores in which the nucleus was drated in a graded acetone series for 45 s each time located eccentrically to centrally (Fig. 3). and then twice in 100% acetone for 45 s each time. Histopathologic examination revealed massive Dehydrated samples were then infiltrated in a graded aggregates of tiny spores (<2.0 µm) interspersed acetone–Spurr’s resin series (at 250 W) for 3 min under within the myosepta and located between muscle bun- vacuum and embedded in 100% Spurr’s resin, then dles (Fig. 4). Each spore had a basophilic-staining polymerized at 60°C for 2 d. Cured sample resin blocks nucleus (in H&E). In longitudinally sectioned skeletal were trimmed with an EM TRIM specimen trimmer muscle, vacuoles (ca. 5 µm diameter) were commonly (Leica Microsystems). Sections were cut with an Ultra- found adjacent to the necrotic skeletal muscle and Cut R ultramicrotome (Leica Miscrosystems) and ultra- among the masses of spores (Figs. 4 & 5). The skeletal thin sections were collected on 200 mesh copper grids. muscle showed both liquefactive and coagulative Ultrathin sections were post-stained with 2% aqueous necrosis (Figs. 4 & 5). Coagulative necrosis tended to uranyl acetate and Reynold’s lead citrate and exam- be accompanied by karyolysis of the myocyte nuclei ined with a H-7000 transmission electron microscope (Fig. 4). In contrast, the myocytes in the remnants of (Hitachi High Tech- nologies America) operated at muscle fibrils contained karyorrhexic and pyknotic 75 kV. Digital images were acquired with a camera nuclei and occasionally stained lightly PAS-positive (MegaViewIII, Soft Imaging Solutions). (Fig. 5). These characteristics, in addition to the pres- ence of pooled proteinaceous fluid, were indicative of liquefactive necrosis (Fig. 4). However, because the tis- RESULTS sue samples were not fixed immediately following the lobsters’ deaths, postmortem tissue degradation must The length of each spiny lobster’s abdomen from be considered in addition to actual pathologic changes. anterior of the first segment to the posterior end of the Spore aggregates located among the necrotic muscle telson was 196 mm (Jupiter specimen) and 144 mm affected by liquefactive necrosis stained lightly (Pompano specimen). On gross examination, the skele- basophilic (Fig. 4). Presumably these spore cells under- tal muscle was totally white and had a cotton-like went degeneration or necrosis.

Fig. 2. Panulirus argus. Specimen from the Jupiter site; Fig. 3. Panulirus argus. Specimen from the Jupiter site; photomicrograph of the skeletal muscle tissue wet mount photomicrograph of the skeletal muscle smear stained with showing numerous microsporidia spores Diff-Quick. Nucleus of the spores located eccentric to central 240 Dis Aquat Org 84: 237–242, 2009

Ultrastructurally, spores and sporoblasts were found sisted of external microvilli (Fig. 6) surrounding the freely within the sarcoplasm of the muscle cells. exospore (approximately 0.02 µm in thickness), which, Mature spores were ovoid to pyriform, with a broad- in turn, covered the endospore (ca. 0.02 µm in thick- ened base (Fig. 6A; mean width ± SD 1.0 ± 0.13 µm, ness). Within the sporoplasm, a single nucleus was range 0.8 to 1.4 µm; mean length ± SD 1.4 ± 0.11 µm, found at the posterior pole, and the polaroplast and range 1.2 to 1.6 µm; N = 16). The spore structure con- anchoring disc were located at the anterior pole (Fig. 6A). The isofilar polar filament had 7 to 8 turns (Fig. 6). TEM sections consisted mostly of mature spores, but some regions of the muscle contained high concentrations of sporoblasts undergoing cytokinesis (Fig. 7).

DISCUSSION

The lobsters examined in this study appeared to be in an advanced stage of microsporidiosis based upon the grossly visi- ble extensive infestation of the abdominal muscle, the highly abundant spores detected through histopathology and the dominant presence of mature spores. Invertebrates often undergo postmortem autolysis or liquefactive necrosis, whereas coagulative necrosis is often referred to as a ‘cooked’ appearance that retains the cellular Fig. 4. Panulirus argus. Specimen from the Jupiter site. Histological section architecture, as defined in general pathology of the skeletal muscle showing numerous microsporidia spores (sp) sur- nomenclature (Jones & Hunt 1983, Roberts rounding coagulative (CN) and liquefactive necrosis (LN) of the skeletal 1989, Couch & Fournie 1993). Liquefactive muscle (H&E, JB-4 section). Vacuoles (V) were commonly found adjacent to the necrotic skeletal muscle tissues among the mass of spores. Karyor- necrosis of the skeletal muscle due to exten- rhexic (white arrowhead) and pyknotic nuclei (arrows) of the myocytes sive microsporidian infection has been were hallmarks of necrosis of the skeletal muscle reported in other crustaceans, such as the freshwater tenuimanus (Langdon 1991) and blue (Findley et al. 1981). Coagulative skeletal muscle necrosis has also been found during the acute phase of infectious myo- necrosis virus (IMNV) syndrome in cultured Pacific white shrimp Litopenaeus vannamei (Lightner et al. 2004, Poulos & Lightner 2006). Shrimp in an advanced chronic phase of myonecrosis progress from coagulative to liq- uefactive necrosis (Lightner et al. 2004). In spite of the massive number of spores invad- ing the skeletal muscle tissues in our speci- mens, coagulative necrosis was observed (recognized as gross changes in the muscle color from translucent gray to white). We speculate that microsporidian spores may produce compounds that suppress lysis and effectively ‘fix’ the tissue, resulting in a char- acteristic ‘cooked’ appearance. Stephens et Fig. 5. Panulirus argus. Specimen from the Jupiter site. Histological section of the skeletal muscle with numerous microsporidia spores (sp) (PAS-MY, al. (2003) described microsporidiosis lesions JB-4 section). Note PAS-positive necrotic muscle tissues (arrows). in the western rock lobster V: vacuoles in as resembling cooked muscle. Kiryu et al.: Microsporidiosis in Panulirus argus 241

(Dennis & Munday 1994). Dennis & Munday (1994) proposed that the microsporidian parasites from these palinurid lobsters be identified as Ame- son sp. (Sprague & Couch 1971) based on key morphological characteristics. Other cases of microsporidiosis in deca- pod crustaceans that have been attrib- uted to Ameson include those affecting the penaeid shrimp Parapenaeus lon- girostris (Campillo & Comps 1977) and the portunid Carcinus maenas, C. mediterraneus (Vivarès & Sprague 1979), (Vivarès & Aze- vedo 1988), Callinectes sapidus (Find- ley et al. 1981) and Portunus pelagicus (Shields & Wood 1991). A spore size of 1.2 × 2.0 µm was documented for A. nelsoni from penaeid shrimp (Lightner Fig. 6. Transmission electron micrographs of mature single microsporidian spore. (A) Specimen from Jupiter site: longitudinally sectioned spore showing 1996). Based on morphological charac- microvilli (MV), nucleus (Nu), polar filament (PF), polaroplast (PP) and teristics described for Ameson, includ- anchoring disc (AD). (B) Specimen from Pompano Beach site: cross-sectioned ing microvilli extending from the sur- spore showing the MV, exospore (white arrow), endospore (black arrow), face of the exospore, a unikaryotic PF and PP spore and an isofilar polar filament (Shields & Wood 1991, Dennis & Mun- day 1994, Lightner 1996), the micro- In Australia, the ornate lobster sporidian identified from our lobster specimens is and western rock lobster infected with microsporidia most probably a member of this genus. had spores measuring 1.4 to 1.8 µm by 2.0 to 2.4 µm The microsporidian species causing the infection (measured by TEM) and in the range of 1.0 to 2.0 described here in Panulirus argus remains to be con- µm (measured by light microscopy), respectively clusively identified. Along with the morphological fea- tures, molecular-based techniques must be used to identify this parasite in future work. Potential risks to the lobster fishery are also currently unknown, but are being investigated. The Caribbean spiny lobster fishery is one of the most valuable fisheries in the Florida and throughout the Caribbean (Hunt 2000, FAO 2001, 2004), so determining the potential for significant effects on this lucrative indus- try is of the utmost importance.

Acknowledgements. This research was sup- ported by Florida State funds under the Fish and Wildlife Health program. Special thanks to D. Dickinson and D. Austin, who sent the spec- imens for examination to FWRI and University of Florida, respectively. M. Bakenhaster, C. Brown and A. Long at FWRI assisted with spec- imen examination and preparation. We thank N. Perry, M. Tabuchi and S. Leslie for their Fig. 7. Transmission electron micrograph of a concentration of mature spores technical assistance in processing histology and dividing sporoblasts in specimen from the Pompano Beach site. Sporob- slides, and Y. Walters, B. Kang and K. Kelley, last (SB), mature spore (MS), anchoring disc (A), developing polar filament who processed tissues for transmission elec- (black arrow) and sites of sporoblast cytokinesis (within black circles) tron microscopy. 242 Dis Aquat Org 84: 237–242, 2009

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Editorial responsibility: Ken Hasson, Submitted: December 22, 2008; Accepted: February 12, 2009 College Station, Texas, USA Proofs received from author(s): April 10, 2009